1. Field of the Invention
This invention relates to isolated nucleotide sequences which are derived from a newly identified immediate early gene of white spot syndrome virus (WSSV) and which have promoter activity to drive the transcription of a target gene in a non-native host cell, thus having great potential for use in the field of biotechnology.
2. Description of the Related Art
The production of recombinant polypeptides/proteins is a very important genetic engineering technique in the field of biotechnology. The basic principle involves the cloning of a target gene capable of expressing a desired gene product (e.g., ribozymes and RNA transcripts, industrial and agricultural enzymes, therapeutic proteins, interferons, interleukins, hormones, growth hormones, antigenic polypeptides, antibodies and the like) into a suitable vector, and the subsequent transfer of the resultant recombinant vector into a competent host cell. The thus-formed recombinant host cell can be cultivated in a suitable culture medium under suitable culture conditions, and expression of the target gene can be induced at an opportune time so as to achieve the object of massive production of the desired gene product.
According to current knowledge and techniques in the field of genetic engineering, Escherichia coli cells are the most widely used and the most effective host cells, and many types of plasmid vectors for harboring in this bacterial species have been developed. Such plasmid vectors are generally constructed to have an inducible artificial promoter cloned upstream of a target gene, so as to control the expression of said target gene. In general, the most commonly used artificial promoters include lac-trp-tac-trc-araBAD-λPRPL, and T7 promoters, and these promoters may be induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG), lactose, arabinose, a change in temperature or the like (S. C. Makrides et al. (1996), Microbiol. Rev., 60: 512-538).
On the other hand, a cloned target gene can be directly cloned into a vector if it contains a constitutive promoter. When such a target gene is used in the construction of a recombinant vector, it is normally unnecessary to elicit the production of recombinant polypeptides/proteins by induction methods.
Other prokaryotic cells that may be used for conducting vector transformation include, but are not limited to, cells derived from: other species/genera of bacteria (such as Bacillus subtilis, Serratia marcescens, Lactobacillus sp., Streptomyces sp. and Salmonella typhi), Cyanobacteria, Actinomycetes and so forth.
However, prokaryotic cells do not, and yeasts only limitedly, carry out post-translational modifications of the expressed polypeptides/proteins, whereas higher eukaryotic cells are able to perform sophisticated protein modifications which are often necessary for the proper function of proteins. Therefore, if post-translational modification(s) is/are needed for recombinant polypeptides/proteins, it may be more desirable to produce the same using eukaryotic cells.
Eukaryotic cells suitable for conducting vector transformation include, for example, fungal cells, protozoan cells, plant cells, insect cells, animal cells, and human cells. Examples of suitable fungal cells are yeast cells, e.g. cells of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces, such as K. lactis and K. marxianus. Suitable plant cells are those derived from gynosperms or angiosperms, preferably monocots and dicots, in particular crops, are derived from the roots, shoots, leaves or meristems of these plants, and are cultured in the form of protoplasts or calli. Examples of suitable insect cells are Drosophila S2 cells, Sf21 cells and Sf9 cells derived from Spodoptera frugiperda, etc. Suitable animal cells may be cultured cells or cells in vivo, preferably derived from vertebrates, and more preferably mammals, and are derived from organs/tissues, such as kidney, liver, lung, ovary, breast, skin, skeleton and blood, of these animals. Representative examples of animal cells include CHO, COS, BHK, HEK-293, HeLa, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562, HepG2, etc.
Vectors for use in transforming the above-indicated host cells include those commonly used in genetic engineering technology, e.g. bacteriophages, such as λ phage; plasmids, such as plasmids from E. coli, including pBR322, pBR325, pUC12, pUC13, pQE-30, pET12, pET30, etc., plasmids from Bacillus subtilis, including pUB110, pTP5, pC194, etc., shuttle vectors for E. coli and Bacillus subtilis, e.g. pHY300PLK, and plasmids from yeasts, including pSH19, pSH15, etc.; cosmids; viruses, including insect viruses, e.g. baculoviruses, animal viruses, e.g. vaccinia viruses, cytomegalovirus (CMV), retroviruses, etc.
To achieve an efficient expression of a selected target gene, it is desirable to construct a vector containing a functional promoter/regulatory sequence(s) operatively connected to a target gene carried by said vector. Such promoter/regulatory sequence(s) may be derived from any one of the following: viruses, bacterial cells, yeast cells, fungal cells, algal cells, plant cells, insect cells, animal cells, and human cells. For example, a promoter useful in E. coli cells includes, but is not limited to, tac promoter, T5 promoter, T7 promoter, T7 A1 promoter, lac promoter, trp promoter, trc promoter, recA promoter, lpp promoter, araBAD promoter, and λPRPL promoter. A promoter useful in the cells of Bacillus sp. includes, but is not limited to, SPO1 promoter, SPO promoter, penP promoter, etc. A promoter useful in yeast cells includes, but is not limited to, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. A promoter useful in insect cells includes, but is not limited to, polyhedrin promoter, P10 promoter, OplE2 (OpMNPV ie2) promoter, etc. A promoter useful in animal cells includes, but is not limited to, SR α promoter, SV40 early promoter, RSV-promoter, HIV-LTR promoter, HSV-TK promoter, CMV promoter, CMV-HSV thymidine kinase promoter, etc. A promoter useful in plant cells includes, e.g., 35S CaMV promoter, actin promoter, ubiquitin promoter, etc. Regulatory elements suitable for use in mammalian cells include CMV-HSV thymidine kinase promoters, SV40 early promoter, RSV-promoters, CMV enhancers and SV40 enhancers.
Further, representative RNA polymerase promoters may be classified into the following types: inducible promoters, constitutive promoters, tissue-specific promoters, and synthetic promoters. Representative inducible promoters are the heat-inducible Hsp70 promoter, a metallothionein promoter, an alcohol dehydrogenase promoter and a galactose promoter; representative constitutive promoters are the Rous sarcoma virus (RSV) LTR promoter, the human cytomegalovirus (CMV) major immediate early gene promoter and the SV40 early promoter; and representative tissue-specific promoters are an alpha globin promoter and a beta globin promoter. Synthetic promoters can be produced by chemically or recombinantly modifying native promoters.
In addition to the lack of post-translational modification machinery, there are other problems associated with expressing some proteins in prokaryotic cells. For example, some expressed heterologous proteins are deposited as insoluble inclusion bodies in prokaryotic cells, making recovery of the proteins difficult. Many of the difficulties associated with prokaryotic expression systems may be overcome by using transformed mammalian cell culture systems to produce post-translationally processed proteins. However, mammalian cell cultures may be relatively inefficient because they grow slowly and are difficult and costly to maintain.
Advances in the culture of insect cells, and the development of baculovirus-based expression systems, have facilitated the expression of heterologous proteins by transformed insect cell lines (Luckow and Summers (1988), Bio/Tech., 6, 47-55; Miller (1988), Annu. Rev. Microbiol., 42, 177-199). To date, the expression of heterologous proteins in transformed insect cell lines has been accomplished primarily using vectors derived from the baculovirus Autographa californica multicapsid nucleopolyhedrosis virus (AcMNPV)(Luckow and Summers (1988), supra; Miller (1988), supra).
Baculoviruses are double-stranded DNA viruses that kill infected insect cells by lysis at the end of a typical infection cycle. A variety of baculoviruses are known, each of which is endemic to a particular arthropod species. Baculoviruses are not known to undergo replication in animals outside the Arthropoda.
Gene expression during natural baculovirus infection of an insect is highly regulated and occurs as an ordered cascade. The viral genes may be classified into four different groups according to their place in this cascade of gene expression: immediate early (IE), delayed early (DE), late, and very late. Early gene expression occurs before the onset of viral DNA replication and appears to be essential for the induction of late viral gene expression (Blissard and Rohrmann(1990), Annu. Rev Entomol., 35: 127-155; Guarino and Summers(1988), J. Virol., 62: 463-471; Miller et al.(1983), Virology, 126: 376-380). Experimental evidence indicates that baculovirus ie genes are transcribed by host RNA polymerase II in the absence of other viral factors. Baculovirus ie genes are therefore understood to have promoters that are recognized by the host cell transcription machinery.
The above descriptions in connection with baculoviruses and ie genes thereof are excerpted from U.S. 20020116723 A1, which discloses the use of promoters derived from a baculovirus immediate early promoter to control expression of a selectable marker gene that confers resistance to one of the family of bleomycin/phleomycin-type antibiotics. Specifically, it is disclosed in U.S. 20020116723 A1 that ie1 and ie2 promoters derived from the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) ie1 and ie2 genes may be operably linked to a selectable marker gene to control transcription from the selectable marker gene, and that the selectable marker gene may be the Streptoalloteichus hindustanus ble gene which confers Zeocin resistance on insect cells.
Patents and published patent applications describing the construction of virus vectors and/or vectors containing viral promoters include, but are not limited to: U.S. Pat. Nos. 5,077,214; 5,162,222, 5,168,062, 5,385,839, US 20020116723 A1, US 20030108524 A1, US 20030108863A1, US 20030229046 A1, US 20040082531 A1, US 20040161841A1, US 20040197313 A1, WO 99/61636 A1, and WO 0105992 A1.
Literature references relevant to the identification of virus promoters and/or regulatory sequences for constructing vectors useful in the transformation and/or transfection of host cells include, but are not limited to: Tom A. Pfeifer et al. (1997), Gene, 788, 183-190; Steven S. Pullen and Paul. D. Friesen, June 1995, 69 (6), 3575-3583; Fan Xiu Zhu et al., J. Virol., July 1999, 73 (7), 5556-5567; R. L. Harrison and B. C. Bonning (2003), J. Gen. Viral., 84 (Pt 7), 1827-1842; E. B. Carstens et al., Virus research (2002), 83, 13-30; M. K Barnhart et al., J. Virol., January 1997, 71 (1), 337-344; L. A. Guarino and M. D. Summers, J. Virol., February 1986, 57 (2), 563-571; V. A. Olson et al., J. Virol., May 2003, 77 (10), 5668-5677; E. A. van Strien et al., Arch Virol. (2000), 145, 2115-2133; V. A. Olson et al., J. Virol., September 2002, 76 (18), 9505-9515; Andrew K Cheung (1999), Nucleic Acid Research, 17 (12), 4637-4646; Alejandra Garcia-Maruniak et al., J, Virol., July 2004, 78 (13), 7036-7051; K. Kojima et al. (2001), Arch Virol., 146, 1407-1414.
In spite of the aforesaid, researchers in the art are still endeavoring to explore any potential promoter and/or regulatory sequence that may be used in the construction of recombinant expression vectors useful in the production of recombinant polypeptides/proteins.
White spot syndrome virus (WSSV) or white spot bacilliform virus (WSBV), which is an enveloped, ellipsoid, large, double stranded DNA virus, is one of the most virulent and hazardous viral pathogens of cultivated shrimps worldwide (K. Inouye et al., Fish Pathol. (1994), 29:149-158 and Fish Pathol. (1996), 31: 39-45; Nakano et al., Fish Pathol. (1994), 29 (2):135-139; Takahashi et al., Fish Pathol. (1994), 29 (2):121-125; H.-Y. Chou et al. (1995), Dis. Aquat. Org., 23:165-173; J. Huang et al., Marine Fish Res. (1995), 16:1-10 and Marine Fish Res. (1995), 16:11-23; C.-F. Lo, et al., Dis. Aquat. Org. (1996), 27, 215-225 and J. Fish. Soc. Taiwan (2003), 30, 1-13; C.-H. Wang et al. (1995), Dis Aquat. Org., 23: 239-242, C. Wongteerasupaya et al. (1995), Dis. Aquat. Org., 21: 69-77; T. W. Flegel (1997), World J. Microbiol. Biotech., 13, 433-442; Y. Lu et al. (1997), J. Gen. Virol., 84:1517-1523). It also attacks many other crustaceans such as crabs and crayfishes. In addition, due to the uniqueness of WSSV, it is difficult to interpret the infection strategy of WSSV by directly applying the infection models of other viruses. As a consequence, the infection strategy of WSSV may need to be investigated ab initio.
Morphologically, the virion of WSSV is a nonoccluded, enveloped particle of approximately 275 by 120 nm with an olive-to-bacilliform shape, and has a nucleocapsid (300 by 70 nm) with periodic striations perpendicular to the long axis (C.-H. Wang et al. (1995), Dis Aquat. Org., 23: 239-242; C. Wongteerasupaya et al. (1995), Dis. Aquat. Org., 21: 69-77). The most prominent feature of WSSV is the presence of a tail-like extension at one end of the virion (Wongteerasupaya et al. (1995), supra; S. Durand et al. (1997), Dis. Aquat. Org., 29:205-211).
Complete genome sequencing has been performed on three WSSV isolates (for Taiwan isolate WSSV T-1, see NCBI Accession No. AF440570; for Thailand isolate, see NCBI Accession No. AF369029; and for China isolate, see NCBI Accession No. AF332093). The WSSV genome (˜300 kb) is ˜30 kb smaller than the 335,593 bp genome of the Ectocarpus siliculosus virus (EsV-1; family Phycodnaviridae), which is the largest virus genome sequenced to date (J. L. van Etten et al. (2002), Arch Virol., 147, 1479-516).
Previous studies on individual genes and analyses of the complete genome sequence suggest that WSSV does not belong to any known virus family (M.-F. Tsai et al., Virology (2000), 277, 92-99 and Virology (2000), 277:100-110; W. J. Liu et al., (2001), Virology 289: 362-377; Feng Yang et al., J. Virol., December 2001, 75 (23): 11811-11820; C. W. Mariëlle et al., Virology. Jul. 20, 2001, 286 (1):7-22; L.-L. Chen et al. (2002), Virology 301: 136-147; H. Marks et al. (2003), J Gen Virol 84:1517-1523). Recently, WSSV has been proposed as the type species of the genus Whispovirus, family Nimaviridae (M. A. Mayo (2002), Arch. Virol., 147, 1655-1663).
In the Applicants' earlier genomic analysis directed to the Taiwan isolate using microarray technique, this isolate was identified to have a total of 532 putative open reading frames (ORFs) that start with an ATG initiation codon and probably encodes a polypeptide of at least 60 amino acids long, amongst which, 39 ORFs have so far been identified as WSSV structural genes and less than a dozen as non-structural genes. In addition, transcripts have been detected for ˜90% of these ORFs (H.-C. Wang, et al. “DNA microarrays of the white spot syndrome virus genome: genes expressed in the gills of infected shrimp,” Marine Biotechnology, in press). In addition, most of the ORFs posted for the Taiwan isolate show no significant similarity to other known proteins based on homology searches against the NCBlnr database. Similar results have been reported for the other two isolates (Mariëlle C. W. van Hulten et al., Virology. Jul. 20, 2001, 286 (1):7-22; Feng Yang et al., J. Virol., December 2001, 75 (23): 11811-11820).
However, although the temporal expression of WSSV genes has been investigated both by individual gene studies (L.-L. Chen et al. (2002), Virology, 301, 136-147; J.-H. Leu, et al. (2005), J. Virol., January 2005, 79 (1), 140-149, W.-J. Liu, et al. (2001), Virology 289, 362-377; M.-F. Tsai et al., Virology (2000), 277, 92-99 and Virology 277 (2000), 100-110) and by global analysis (M.-F. Tsai et al. (2004), J. Virol. 78, 11360-11370; H.-C. Wang et al., “DNA microarrays of the white spot syndrome virus genome: genes expressed in the gills of infected shrimp,” Marine Biotechnology, in press), heretofore, no WSSV immediate early (IE) gene has been identified.
As noted from literature, the expression of viral IE genes depends on the host cell machinery and occurs independently of any viral de novo protein synthesis, which means that the IE genes are especially important in determining host range (P. D. Friesen (1997), “Regulation of Baculovirus early gene expression, ” In: Miller, L. K., (Ed.), The baculoviruses. Plenum Press, New York and London, pp. 141-170). The IE gene products, once expressed, may function as regulatory trans-acting factors and may serve to initiate viral replicative events during infection. In the cascade of viral regulatory events, successive stages of virus replication are dependent on the proper expression of the genes in the preceding stage. For example, during infection by the large DNA viruses, such as baculoviruses and herpesviruses, gene expression is regulated such that the immediate early (IE or α) genes are transcribed first, followed by the expression of the early (E or β) and late (L or γ) genes, respectively (G. W. Blissard (1996), Cytotechnology, 20, 73-93, G. W. Blissard and G. F. Rohrmann (1990), Annu. Rev. Entomol., 35, 127-155; P. D. Friesen and L. K Miller (1986), Curr. Top. Microbiol. Immunol. 131, 31-49; R. W. Honess and B. Roizman (1974), J. Virol., 14, 8-19).
To study the transcription of viral IE genes, viral infection is induced in the presence of a protein synthesis inhibitor, usually cycloheximide (CHX), which prevents de novo protein synthesis by preventing translation. In case that translation (but not transcription) of the IE genes is impeded, the viral infection cycle will likewise be blocked at the IE stage. Therefore, the detected presence of RNA transcript during viral infection in the constant presence of CHX is good evidence for the identification of viral IE genes.
Here for the first time, in spite of the lack of any well-acknowledged immortalized shrimp cell line and the difficulty of using CHX in vivo, the Applicants successfully used CHX as an inhibitor to block de nova viral protein synthesis. A global analysis microarray technique and RT-PCR was subsequently used to determine the transcription pattern of WSSV, from the results of which 3 candidate WSSV immediate early (ie) genes were identified and were designated as ie1, ie2 and ie3. In addition, promoter-regulatory regions cloned from the WSSV ie1 gene were proven to have promoter activity in non-native host cells, i.e. Sf9 insect cells, thus having great potential for use in the field of recombinant DNA technology.